Magnetic beads having surface glycoconjugates and use thereof

ABSTRACT

Magnetic beads that include polyvalent ligands comprising various carbohydrates are described. Methods for fabricating such magnetic beads are also provided as well as methods of their use to capture and enrich pathogen cell population for subsequent culture, lysis and identification.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims priority under 35 U.S.C. §119(e) toUnited States Provisional patent application, U.S. Ser. No. 61/375,320,filed Aug. 20, 2010, the entire contents of which are incorporatedherein by reference.

FIELD OF THE DISCLOSURE

This disclosure relates generally to compositions, systems and methodsfor detecting biological target analytes (such as microorganismsincluding bacteria, fungi and viruses) in a sample. More specifically,this disclosure relates to magnetic beads having surfaceglycoconjugates, methods of making such beads and to methods of usethereof to capture microorganism(s) (such as bacteria, fungi, viruses)from samples for detection and identification.

BACKGROUND

Food companies are required to test for presence of common foodpathogens such as E. coli 0157:H7, Listeria, Salmonella, Campylobacter,Vibrio, etc. to protecting public health. Tests for detecting foodpathogens are also performed by health service labs and governmentagencies to monitor and track outbreaks of food poisoning. Reducing thetime necessary to obtain results of food testing is important for thefood processing industry as it reduces the time and costs associatedwith storage of food prior to delivery till food testing results areobtained.

The most common method for identifying the presence of microorganisms isby enriching in selective broths and platting on defined agars.Classical platting methods require 3-5 days for confirmation (sometimeslonger depending on the organism), and advanced skills in microbiology.The largest unmet need within the food testing market is the ability toproduce results in one work shift, which is typically defined as eighthours or less.

Molecular methods such as PCR currently play a small role in testingpresence of microorganisms. Real-time PCR is valuable because itcombines simplicity with specificity and sensitivity. PCR, however, hasits limitations, due to the necessity of sample preparation which can betime-consuming, and is technically challenging and expensive in view ofthe existence of a wide variety of food samples having differentchemical and physical properties, and the necessity to process verylarge sample volumes. In addition many foods, such as meat products,contain PCR inhibitors.

One solution for this problem relies on magnetic properties. Forexample, magnetic seeds (magnetite) were used to capture single cellorganisms in the presence of a calcium chloride binder, as described inU.S. Pat. No. 4,001,197. The magnetic beads can be DYNABEADS (Dynal AS,Oslo, Norway) functionalized to have positively or negatively charged,hydrophilic or hydrophobic surfaces, as taught in U.S. Pat. No.7,560,228. Pathogen cells can also be captured by non-specificadsorption on the surfaces of magnetic beads (BUGS'n BEADS™ from NorDiagInc., West Chester, Pa., US).

The above mentioned techniques rely on non-specific adsorption that mayalso bind various types of proteins that can inhibit and/or interferewith subsequent PCR reactions. The inhibition/interference can beexacerbated by subsequent in situ lysis and PCR in the presence of theseproteins-bound magnetic beads.

Bacterial cellular surfaces comprise a variety of complex carbohydratestructures, such as glycoproteins, glycolipids, glycosaminoglycans, andproteoglycans. These glycoconjugates play a central role in cell-to-celladhesion and subsequent recognition and receptor activation, asdiscussed in G. M. Whitesides et al., Angew. Chem. Int. Ed. 1998, 37,2754-2794. And yet, the surfaces of different bacterial species arechemically and morphologically quite distinct.

Certain cells are able to selectively bind to one particularglycoconjugate but not the others. In practical applications, pathogencells can be captured with magnetic beads having carbohydrates,including monosaccharides, disaccharides, oligosaccharides, andpolysaccharides, immobilized on the bead surfaces, as described in thepublished U.S. Patent Application No. 2009/0186346A1. The binding of apathogen cell onto a carbohydrate-modified magnetic bead is “monovalent”as schematically shown on FIG. 1.

The affinity, efficiency and binding strength of the structure depictedon FIG. 1 are weak. It may not withstand repetitive washing and rinsingto remove of debris and undesirable materials from the biologicalsample. Accordingly, better methods have to be employed to allow thoseskilled in the art to solve one or more of the above-mentioned problems.

SUMMARY OF DISCLOSURE

The present application, in some embodiments, describes compositionsoperable for “polyvalent” binding to cells—such as, but not limited to,cells of microorganisms (including bacteria, fungi and viruses); methodsof synthesizing these compositions and methods of using compositions ofthe disclosure to detect cells present in a sample. In some embodiments,compositions of the disclosure may be used in methods for food testingfor rapid, robust and cost-effective capture and subsequent analysis ofmicroorganisms present in food samples.

In some embodiments, the present disclosure describes compositions,comprising: a paramagnetic bead; a plurality of hydrophilic copolymerbridges, each bridge being covalently bonded to the paramagnetic bead;and a plurality of carbohydrates, each carbohydrate being covalentlybonded to the same or different hydrophilic copolymer bridge by forminga glycoconjugate with the respective hydrophilic copolymer.

In some embodiments, compositions of the disclosure may have thestructure:

MB-(HP)_(n)-(S)_(m),

wherein MB is a magnetic bead; HP is hydrophilic polymer bridge; S is acarbohydrate; and each of n and m is an integer, and wherein n≧1 andm≧1, with the further proviso that if n=1, then m≧2.

In some embodiments, a composition of the disclosure may have eachhydrophilic polymer bridge (HP) bonded to a paramagnetic bead (MB) via alink formed by an amino group. In some embodiments a paramagnetic bead(MB) may be a bead described by DYNABEADS®.

In some embodiments, each hydrophilic copolymer may be the same in acomposition of the disclosure. In some embodiments, at least onehydrophilic copolymer is different from at least one other hydrophiliccopolymer, in a composition of the disclosure.

In some embodiments, hydrophilic copolymer bridges are formed offunctionalized hydrophilic copolymers and may be functionalizedacrylates, poly(alkylene glycols), alkoxy poly(alkylene glycols),copolymers of methylvinyl ether and maleic acid, urethanes,ethyleneimines, polyurethane-polyether copolymers, copolymers havingunits derived from vinyl alcohol, N-vinyl lactams, vinyl pyrrolidone,amides, maleic anhydride, styrenesulfonates, vinylsulfonic acid,vinylsulfonates, N-vinylamides or 3-hydroxybutyric acid, and variouscombinations and derivatives thereof.

In some embodiments, a composition of the disclosure may comprisefunctionalized acrylates such as but not limited to functionalizedcopolymers having units derived from acrylic acid, methacrylic acid,2-hydroxyalkyl acrylate, 2-hydroxyalkylmethacrylate, acrylamides,methacrylamides, epoxy-acrylates, and combinations and derivativesthereof.

In some embodiments, each of the functionalized hydrophilic copolymers,in a composition of the disclosure, may comprise a plurality offunctional groups, comprising: a first functional group to form acovalent linkage between a magnetic bead and a respective hydrophiliccopolymer bridge; and at least one second functional group, to attach atleast one carbohydrate to the hydrophilic copolymer by formingglycoconjugate(s) between the carbohydrate(s) and the hydrophiliccopolymer. In some embodiments, the first functional group and thesecond functional group may be the same functional group. In someembodiments, in a plurality of functional groups at least one functionalgroup is different from at least one other functional group. Exemplarynon limiting functional groups may include without limitation: an amino,a hydroxyl, a carboxyl, a N-hydroxysuccinimide, an ester ofpentafluorophenol, a maleimide, an epoxy, an aldehyde, a ketone, acyanuryl, a pyrrolidinedione, an alkyne and/or an azide and/orcombinations of these groups.

In some embodiments, a composition of the disclosure may comprisecarbohydrates comprising monosaccharides, disaccharides, trisaccharides,tetrasaccharides, oligosaccahrides, polysaccarides or N-modifiedderivatives thereof. In some exemplary embodiments, monosaccharides in acomposition of the disclosure may be glucose, galactose, fructose,mannose, lyxose and xylose and/or N-modified derivatives thereof. Insome exemplary embodiments, disaccharides in a composition of thedisclosure may be sucrose, lactose, maltose, isomaltose, lactulose,trehalose and/or N-modified derivatives thereof. In some exemplaryembodiments, polysaccharides in a composition of the disclosure may becellulose, glycan, dextrin, amylase, amylopectine and/or N-modifiedderivatives thereof.

In some exemplary embodiments, carbohydrates in a composition of thedisclosure may be a sialic acid, amine-containing saccharides,saccharide conjugates of glycans, saccharide conjugates ofaminocyclitols and/or N-modified derivatives thereof.

In some embodiments, a composition of the disclosure may comprise thesame carbohydrate for each carbohydrate moiety. In some embodiments, atleast one carbohydrate is different from at least one othercarbohydrate, in a composition of the disclosure.

In some embodiments, a composition of the disclosure may comprisehydrophilic copolymers where each hydrophilic copolymer has theHildebrand solubility parameter δ of at least about 10 MPa^(1/2). Insome embodiments, a composition of the disclosure may comprisehydrophilic copolymers wherein the Hildebrand solubility parameter δ isat least about 20 MPa^(1/2).

In some embodiments, a composition of the disclosure may comprisehydrophilic copolymers wherein the Hildebrand solubility parameter δ isat least about 25 MPa^(1/2).

In some embodiments, a composition of the disclosure may comprisehydrophilic copolymers each having the weight-averaged molecular weightof between about 5,000 and about 5,000,000 Daltons. In some embodiments,the weight-averaged molecular weight of hydrophilic copolymers incompositions of the disclosure may be between about 50,000 and about1,000,000 Daltons. In some embodiments, the weight-averaged molecularweight of hydrophilic copolymers in compositions of the disclosure maybe between about 100,000 and about 500,000 Daltons.

The disclosure also describes methods of making compositions of thedisclosure. A method for fabricating a paramagnetic bead composition,may comprise: immobilizing a plurality of functionalized hydrophiliccopolymers on the surface of a paramagnetic bead; and bonding at leastone carbohydrate to each of at least two hydrophilic copolymers, whereineach of the functionalized hydrophilic copolymers includes a pluralityof functional groups, comprising a first functional group and at leastone second functional group, to thereby obtain the paramagnetic beadcomposition.

In some embodiments, the step of immobilizing comprises reacting aplurality of functionalized hydrophilic copolymers with a paramagneticbead, wherein a covalent linkage is formed between the paramagnetic beadand each hydrophilic copolymer.

In some embodiments, the step of bonding comprises reacting a pluralityof functionalized hydrophilic copolymers with at least onecarbohydrate(s), wherein at least two glycoconjugates are formed betweenthe at least one carbohydrate and the hydrophilic copolymers. In someembodiments of a method of the disclosure, hydrophilic copolymers may befunctionalized and in non-limiting examples may comprise: functionalizedacrylates, poly(alkylene glycols), alkoxy poly(alkylene glycols),copolymers of methylvinyl ether and maleic acid, urethanes,ethyleneimines, polyurethane-polyether copolymers, and copolymers havingunits derived from vinyl alcohol, N-vinyl lactams, vinyl pyrrolidone,amides, maleic anhydride, styrenesulfonates, vinylsulfonic acid,vinylsulfonates or 3-hydroxybutyric acid, and/or derivatives thereof.

In some example embodiments, functionalized acrylates may comprisefunctionalized copolymers having units derived from acrylic acid,methacrylic acid, 2-hydroxyalkyl acrylate, 2-hydroxyalkylmethacrylate,acrylamides, methacrylamides, epoxy-acrylates, and/or derivativesthereof.

In some embodiments, in the step of immobilizing may comprising reactinga plurality of functionalized hydrophilic copolymers with a paramagneticbead, wherein a covalent linkage is formed between the paramagnetic beadand each hydrophilic copolymer, wherein the plurality of functionalgroups comprises the same functional group(s).

In some embodiments of the method, in the plurality of functional groupsat least one functional group is different from at least one otherfunctional group.

In some embodiments, a functional group may be a hydroxyl, a carboxyl, aN-hydroxysuccinimide (NHS), an epoxy, a cyanuryl, an alkyne and apyrrolidinedione group.

Methods of the disclosure, in some embodiments, may use carbohydratescomprising monosaccharides, disaccharides, trisaccharides,tetrasaccharides, oligosaccharides, polysaccharides and/or N-modifiedderivatives thereof.

Exemplary monosaccharides used may comprise glucose, galactose,fructose, mannose, lyxose and xylose and/or N-modified derivativesthereof. Exemplary disaccharides used may comprise sucrose, lactose,maltose, isomaltose, lactulose, trehalose and/or N-modified derivativesthereof. Exemplary polysaccharides used in methods of the disclosure maycomprise cellulose, glycan, dextrin, amylase, amylopectine andN-modified derivatives thereof.

In some embodiments, methods of the disclosure may use carbohydratessuch as, but not limited to, sialic acid, amine-containing saccharides,saccharide conjugates of glycans, saccharide conjugates ofaminocyclitols and/or N-modified derivatives thereof.

In some embodiments, a method of making a composition of the disclosuremay comprise using carbohydrates wherein at least two carbohydrates arebonded and each individual carbohydrate is the same. In some embodimentsof a method of the disclosure, at least two carbohydrates are bonded andat least one individual carbohydrate is different from at least oneother individual carbohydrate.

The present disclosure also describes methods for capturing a populationof microorganisms from a biological sample, comprising: combining acomposition of the disclosure comprising: a paramagnetic bead; aplurality of hydrophilic copolymer bridges, each bridge being covalentlybonded to the paramagnetic bead; and a plurality of carbohydrates, eachcarbohydrate being covalently bonded to the same or differenthydrophilic copolymer bridge by forming a glycoconjugate with therespective hydrophilic copolymer, with the biological sample for a timeto form composition-microorganism complexes; separating thecomposition-microorganism complexes from the sample under a magneticfield; and collecting the captured microorganisms, wherein thepopulation of microorganisms has binding specificity for thecarbohydrate.

A variety of microorganisms may be detected and/or captured usingcompositions of the disclosure such as bacteria including gram negativeand gram positive species; viruses, fungi, and spores thereof.

In some embodiments of the method of capturing microorganisms, thepopulation of microorganisms captured is a subpopulation ofmicroorganisms present in the biological sample.

In some embodiments of the method, the carbohydrate is mannose and thepopulation of microorganisms has binding specificity for mannose.

A method of capturing microorganisms of the disclosure may additionallycomprising downstream steps for detection and identification of themicrobe. Such steps in non-limiting examples may comprise one or more ofthe following steps: performing a nucleic acid extraction on thecaptured microorganisms; Real-Time PCR analysis, culturing the capturedmicroorganisms to increase the number of microorganisms for furtheranalysis, DNA sequencing, immunoassays and the like.

These and other features of the present disclosure will become betterunderstood with reference to the following description, drawings, andappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described below, are for illustration purposes only, andare not intended to limit the scope of the present disclosure in anyway.

FIG. 1 depicts a scheme showing monovalent interactions between apathogen cell and a carbohydrate-modified magnetic bead;

FIG. 2 schematically shows a magnetic bead with polyvalent carbohydratependants attached to it, according to embodiments of the presentdisclosure;

FIG. 3 schematically shows a potential mechanism of polyvalent bindingof pathogen cells, according to embodiments of the present disclosure;

FIG. 4 schematically shows exemplary ways of using polyvalent magneticbeads, according to embodiments of the present disclosure; and

FIG. 5 shows results of a real-time PCR analysis of E. coli strainAAEC356 recovery by polyvalent mannose magnetic beads prepared accordingto one embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to some embodiments of thedisclosure. While the disclosure will be described in conjunction withthe embodiments discussed below, it will be understood that they are notintended to limit the disclosure to those embodiments. On the contrary,the disclosure is intended to cover alternatives, modifications, andequivalents, which may be included within the disclosure as defined bythe appended claims.

The present disclosure relates to compositions comprising magnetic beadsthat include “polyvalent” ligands comprising a plurality ofcarbohydrates that are operable to capture cell populations in samples(such as microorganism cells) using polyvalent ligand binding.Compositions of the disclosure, methods for making such compositions andmethods of using the present compositions for capturing and/or detectingcells and microbes (such as bacterial cells, viruses and fungi) aredescribed.

According to some embodiments of the disclosure, magnetic beadcompositions are provided, which comprise a paramagnetic bead, aplurality of hydrophilic copolymer bridges, each bridge being covalentlybonded to the paramagnetic bead, and a plurality of carbohydrates, eachcarbohydrate being connected to the same or different hydrophiliccopolymer by forming a glycoconjugate with the respective hydrophiliccopolymer.

According to other embodiments of the disclosure, methods forfabricating paramagnetic bead compositions are provided. Such methodsinclude immobilizing a plurality of functionalized hydrophiliccopolymers on the surface of a paramagnetic bead and bonding at leastone carbohydrate to the hydrophilic polymer, wherein each of thefunctionalized hydrophilic copolymers includes a plurality of functionalgroups, comprising a first functional group and at least one secondfunctional group.

According to yet other embodiments of the disclosure, methods of usingpolyvalent magnetic beads for capturing a microorganism from abiological sample are provided. Such methods include combining aparamagnetic bead composition of the disclosure with a biological samplefor a certain time to form a composition-microorganism complex, removingthe composition-microorganism complex under a magnetic field, andcollecting the captured microorganism, where a paramagnetic beadcomposition of the disclosure includes a paramagnetic bead, a pluralityof hydrophilic copolymer bridges, each bridge being covalently bonded tothe paramagnetic bead, and a plurality of carbohydrates, eachcarbohydrate being connected to the same or different hydrophiliccopolymer by forming a glycoconjugate with the respective hydrophiliccopolymer. A method of the disclosure may further comprise analyzing acaptured microorganism by one or more methods for microorganism andviral analysis such as but not limited to, nucleic acid extraction, DNAextraction, amplification of nucleic acids (e.g., Real-Time PCR and/orTaqMan® Assay), hybridization of nucleic acids, DNA sequencing and thelike. For example, in one embodiment a method may further comprisesperforming a nucleic acid extraction and Real-Time PCR analysis of thecaptured microorganisms. In further embodiments, the method comprisesculturing the captured microorganisms for further analysis.

In other embodiments, a method for capturing a population ofmicroorganisms from a biological sample is provided comprising combininga composition of the disclosure with a biological sample for a time toform composition-microorganism complexes; separating thecomposition-microorganism complexes from the sample under a magneticfield; and collecting the captured microorganisms, wherein thepopulation of microorganisms has binding specificity for thecarbohydrate comprised in the composition of the disclosure. In someembodiments, the population of microorganisms captured may be asubpopulation of microorganisms of the total population ofmicroorganisms present in a biological sample.

In one embodiment, a carbohydrate moiety comprised in a composition ofthe disclosure is mannose and the population of microorganisms capturedby a method of the disclosure, using the composition comprising mannose,has binding specificity for mannose.

Unless stated otherwise, the following terms, definitions, andabbreviations as used herein are intended to have the followingmeanings. Whenever appropriate, terms used in the singular will alsoinclude the plural and vice versa. In the event that any definition setforth below conflicts with the usage of that word in any other document,including any document incorporated herein by reference, the definitionset forth below shall always control for purposes of interpreting thisspecification and its associated claims unless a contrary meaning isclearly intended.

For the purposes of the present disclosure, the term “glycoconjugate”refers to a carbohydrate (such as a saccharide) covalently linked toanother chemical group, e.g., to a polymer. Glycoconjugates containglycosidic linkages, e.g., glycolipids, glycopeptides, glucoproteins orglycosaminoglycans and sialosides.

The term “saccharide” as used herein refers to any molecule comprising asaccharide moiety and encompasses both monosaccharides andpolysaccarides such as di-, tri-, tetra-, oligo-saccharides, etc.“Saccharide” may also be used to refer to biomolecules containingsaccharides, among other moieties. The term “polyvalent glycoconjugate”as used herein refers to glycoconjugates comprising a plurality ofcarbohydrates covalently linked to another chemical group.

The term “monosaccharide” as used herein refers to any carbohydrate (“asimple sugar”), such as triose, tetrose, pentose or hexose (includingboth aldose and ketose forms) that cannot be hydrolyzed to form simplersugars.

The term “polysaccharide” as used herein refers to polymers made up of aplurality of monosaccharide units joined together by glycosidiclinkages.

The term “oligosaccharide” as used herein refers to a polysaccharidecontaining a small number (e.g., three to ten) of saccharide units.

The term “glycoprotein” as used herein refers to a biological moleculecomprising a protein and a carbohydrate covalently linked together.

The term “glycosidic linkage” as used herein refers to a bond formedbetween the hemiacetal group of a saccharide, or of a derivativethereof, and the hydroxyl group of an organic compound, e.g., of analcohol or a glycol.

The terms “magnetic bead” and “magnetic particle” as used herein referto any bead or other solid particulate material, which can be sphericalor irregularly shaped, and which can be attracted by a magnetic field.As used herein, the meanings of the terms “magnetic bead” and “magneticparticle” are identical and they are used interchangeably.

The term “magnetic” for the purposes of the present disclosure means“paramagnetic.” The term “paramagnetic” as used herein refers to amaterial that is not magnetic naturally (i.e., not magnetic in theabsence of the externally applied magnetic field), but when placed insuch an externally applied magnetic field, is magnetized to form its ownmagnetic field, which is parallel to the applied field, to the extentproportional to the applied field.

The term “polyvalent” for the purposes of the present disclosure means“multifunctional” and, accordingly, “polyvalent magnetic bead” refers toa magnetic bead bearing at least one scaffold comprising a hydrophilicpolymer having at least two carbohydrates pendant from the hydrophilicpolymer's chain, or at least two scaffolds each comprising a hydrophilicpolymer having at least one carbohydrate(s) pendant from the hydrophilicpolymers' chains.

The term “monomer,” in accordance with the definition adopted by theInternational Union of Pure and Applied Chemistry (IUPAC), refers to amolecule which is capable of undergoing polymerization therebycontributing constitutional units to the essential structure of amacromolecule (a polymer).

The term “polymer” is defined for the purposes of the present disclosureas being inclusive of copolymers and oligomers. The term “copolymer” isdefined as a polymer derived from more than one species of monomer,including copolymers that are obtained by copolymerization of twomonomer species, those obtained from three monomers species(“terpolymers”), those obtained from four monomers species(“quaterpolymers”), etc. The term “oligomer” is defined as a lowmolecular weight polymer in which the number of repeating units does notexceed twenty.

The term “copolymer” is further defined as being inclusive of randomcopolymers, alternating copolymers, graft copolymers, and blockcopolymers. The term “random copolymer” is defined as a copolymercomprising macromolecules in which the probability of finding a givenmonomeric unit at any given site in the chain is independent of thenature of the adjacent units. In a random copolymer, the sequencedistribution of monomeric units follows Bernoullian statistics. The term“alternating copolymer” is defined as a copolymer comprisingmacromolecules that include two species of monomeric units inalternating sequence. Copolymers of any tacticity are included withinthe term “copolymer.”

The term “hydrophilic polymer” is defined for the purposes of thepresent disclosure as a polymer, which has a range of water solubilityat ambient conditions from water miscible, slightly water soluble, towater swellable.

The term “Hildebrand solubility parameter” is defined as a parameterindicating the cohesive energy density of a substance. The δ parameteris determined as δ=(ΔE/V)^(1/2), wherein δ is Hildebrand solubilityparameter, ΔE is the energy of vaporization, and V is the molar volume.

For the purposes of the present disclosure, “hydrophilic polymer” and“hydrophilic copolymer” are defined as the (co)polymers that exhibitHildebrand solubility parameter δ that is equal to or greater than about10 MPa^(1/2) (for comparison purposes only, acetone exhibits δ=19.7MPa^(1/2)), such as equal to, or greater than, about 20 MPa^(1/2), forexample, equal to, or greater than about 25 MPa^(1/2) (e.g.,polyacrylamide and water exhibit δ of 27 and 48 MPa^(1/2),respectively).

The terms “acrylic,” “polyacrylic,” “acrylate” or “polyacrylate” referto a product that is inclusive of a monomer, oligomer, and pre-polymer,as applicable, having at least one acrylic moiety, CH₂═CH—COO—, ormethacrylic moiety, CH₂═C(CH₃)—COO—, or derived from a product having atleast one acrylic moiety or methacrylic moiety (e.g., polymer bypolymerization of such a product).

The abbreviation “PCR” refers to polymerase chain reaction, which is amethod of producing multiple copies of specific DNA sequences fordetection and evaluation.

The abbreviation “TaqMan® PCR” refers to a kind of PCR that utilizes the5′-3′ nuclease activity of Taq polymerase to cleave a dual-labeled probeduring hybridization to the complementary target sequence andfluorophore-based detection.

As used herein, the term “Ct” represents the PCR cycle number when thesignal is first recorded as statistically significant. The term “Cq”designates quantification cycle and is interchangeable with the term“Ct” (See e.g., “MIQE: Minimum Information for Publication ofQuantitative Real-Time PCR Experiments,” Clinical Chemistry 55:4;611-622 (2009)).

The use of “or” herein means “and/or” unless stated otherwise or wherethe use of “and/or” is clearly inappropriate. The use of “a” hereinmeans “one or more” unless stated otherwise. The use of “comprise,”“comprises,” “comprising,” “include,” “includes” and “including” areinterchangeable and not intended to be limiting. It should also beunderstood that in some embodiments the order of steps or order forperforming certain actions is immaterial so long as the presentteachings remain operable. Moreover, in some embodiments two or moresteps or actions can be conducted simultaneously.

The present disclosure describes compositions having “polyvalent” ligandbinding characteristics. Compositions of the disclosure comprisemagnetic beads or magnetic particles and a plurality of hydrophilicpolymers covalently bonded to such magnetic beads or particles. In someembodiments, compositions of the disclosure also comprise a plurality ofcarbohydrates, each carbohydrate being connected to the same ordifferent hydrophilic copolymer by forming a glycoconjugate with therespective hydrophilic copolymer.

The covalent bonding of the hydrophilic polymers to the magnetic beadsor magnetic particles is achieved by reaction of functional groups thatare present on the surface of the magnetic beads or magnetic particleswith the functional groups that are present in the hydrophilic polymers,as described in detail below. As a result of such a reaction, a covalentlink is formed, which can be can be, for example, via an amide link or asecondary amine link, as described in more detail below. Those havingordinary skill in the art, in light of this specification, will realizethat depending on the nature of the functional groups that are presenton the surface of the beads and the nature of the functional groups thatare present in the hydrophilic polymers, other types of covalent linksmay be formed via which the hydrophilic polymers can be covalentlybonded to the magnetic beads or particles.

The magnetic beads or particles are made of a paramagnetic material andcan be spherical or irregularly shaped. The spherical beads can have adiameter between about 0.1 μm and about 50 μm, such as between about 1μm and about 5 μm. One type of magnetic beads that may be used isDynabeads® available from Dynal AS, Oslo, Norway. Those having ordinaryskill in the art, in light of this specification, can select othersuitable magnetic beads. Some non-limiting examples of other suitablemagnetic beads that may be used for the purpose of covalent binding ofhydrophilic polymer(s) include MagMax™ beads available from AppliedBiosystems, Foster City, Calif., BioMag® beads available fromPolysciences, Inc. of Warrington, Pa. or BcMag™ beads available fromBioClone Inc. of San Diego, Calif., so long as such beads are modifiedto contain the surface functional groups described below.

One embodiment of the disclosure describes magnetic bead compositionscomprising a paramagnetic bead, a plurality of hydrophilic copolymerbridges, each bridge being covalently bonded to the paramagnetic bead,and a plurality of carbohydrates, each carbohydrate being connected tothe same or different hydrophilic copolymer by forming a glycoconjugatewith the respective hydrophilic copolymer. In some embodiments, acomposition of the disclosure as described here may have “polyvalent”ligand binding characteristics. For example, poly(glycoconjugates)compositions of the disclosure may be operable to bind to one or moremolecules on the surface of microorganisms, such as, but not limited to,bacteria, fungi, viruses and spores thereof. Accordingly, compositionsof the disclosure may be used in methods of the disclosure (described indetail below) to capture, enrich and/or detect cell populations, such asmicrobial cell populations from samples that may contain/be contaminatedwith a microbe.

In some embodiments, hydrophilic polymers that are covalently attachedto a magnetic bead may bear at least two carbohydrate residues pendantfrom at least one polymeric chain. A general structure of a compositionof the disclosure can be schematically expressed as structure (A):

MB-(HP)_(n)-(S)_(m),  (A)

wherein “MB” is a magnetic bead, “HP” is hydrophilic polymer, “S” is acarbohydrate (i.e., a sugar), n is an integer indicating the number ofhydrophilic polymeric chains covalently bonded to the magnetic bead, andm is an integer indicating the number of carbohydrate residuesconjugated to each hydrophilic polymer. Hydrophilic polymer(s) serve asbridge(s) connecting a magnetic bead with one or more carbohydrate(s).As discussed in more detail below, each hydrophilic polymer iscovalently bonded to the magnetic bead through its pendant functionalgroups along the polymer chain (see FIG. 2), and is also connected tothe carbohydrate portion by forming a glycoconjugate.

It should be understood that the number of hydrophilic polymeric chainscovalently bonded to the magnetic bead is at least one and the number ofcarbohydrate residues conjugated to each hydrophilic polymer is also atleast one. In other words, in Structure A above, n≧1 and m≧1; however,if n=1, then m≧2.

In some embodiments, the capability of the present compositions forcapturing microorganisms relies on the number and chemical nature ofcarbohydrate molecules along a hydrophilic polymer chain. Those havingordinary skill in the art, in light of this disclosure, can appreciatethe kinetic benefit of having a polyvalent interaction extending awayfrom a magnetic bead/particle, and can determine the number of polymerchains that are to be attached to a magnetic bead.

Among the different variety of hydrophilic polymeric chains, that may beattached to a magnetic bead, individual hydrophilic polymers can be thesame or different, as desired, or some of hydrophilic polymers can bethe same and some can be different, in any combination. One exemplaryembodiment of the architecture of a polyvalent magnetic bead of thepresent disclosure is shown in FIG. 2. In this embodiment, a compositionof the disclosure comprises a paramagnetic bead onto which multiplemobile scaffolds of hydrophilic polymer(s) are attached. These mobilescaffolds of hydrophilic polymer(s) are covalently immobilized on theparamagnetic bead and have carbohydrate pendants along the polymerchains.

In order to enable bonding of hydrophilic polymer(s) to a magnetic bead,the surface of a magnetic bead may be modified to include at least onefunctional group such as amino group. In light of the present teachings,those having ordinary skill in the art, may modify the surface of amagnetic bead in a different way and include functional groups(s) otherthan amino group, if desired. Non-limiting examples of such otherfunctional groups(s) that may be present on the surface of a magneticbead include aldehyde, alkyne, azide, biotin, N,N′-carbonyl diimidazole,carboxylate, epoxy, hydrazide, hydroxyl, iminodiacetic acid, iodoacetyl,NHS, pentadiene, silanol, streptavidin, sulfhydryl, thiol, tosyl,vinylsulfone or any combination thereof.

A mobile scaffold of hydrophilic polymer chains, such as that shown inFIG. 2, enables cell-adhesive sugar pendants to reach out into themedium (e.g., sample, diluted sample), to find binding ligands containedtherein thereby greatly improving the kinetics of the binding reaction.As shown on FIG. 3, when microbes (shown as a pathogen cells) areexposed to compositions comprising polyvalent magnetic beads of thedisclosure, multiple sugar pendants can latch onto the cell surfaces orenvelopes of microbes resulting in multiple binding thereby enabling anapplied magnetic field to extract a bound (captured) microbe from abiological sample.

The cumulative binding force due to multiple carbohydrate recognitionsites is strong enough to withstand subsequent repetitive washing andrinsing to rid of debris and other undesirable materials, for example,proteins and PCR inhibitors. In addition, sugar pendants can bespecially designed to selectively bind one type or strain of organismbut not others. The scaffold polymer is hydrophilic by design such thatit reduces, if not eliminates, non-specific adsorption of proteins andother hydrophobic biomolecules.

Various hydrophilic polymers may be used in compositions of the presentdisclosure. In some embodiments, use of only various functionalizedcopolymers or co-oligomers is contemplated, as described in more detailbelow, including random, alternating, graft and block copolymers, all ofwhich are functionalized. Copolymers can be co-, ter-, quatropolymers,etc., if desired. The use of homopolymers is not contemplated.

The Hildebrand solubility parameter δ of hydrophilic copolymers that maybe used can be at least about 10 MPa^(1/2), such as equal to, or greaterthan, about 20 MPa^(1/2), for example, equal to, or greater than about25 MPa^(1/2). The weight-averaged molecular weight of hydrophiliccopolymers that can be used can be between about 5,000 and 5,000,000Daltons, for example, between about 50,000 and 1,000,000 Daltons, suchas about 100,000 to 500,000 Daltons.

Hydrophilic copolymers comprised in compositions of the disclosureinclude at least two functional groups. A first functional group is toattach a hydrophilic copolymer to a magnetic bead covalently, and asecond functional group (and additional functional group(s)) can be usedto attach carbohydrate(s) to a hydrophilic copolymer by formingglycoconjugate(s) between the carbohydrate(s) and the hydrophiliccopolymer chain(s).

In some embodiments, all the functional groups that are present in afunctionalized hydrophilic copolymer are the same. In other embodiments,at least one of these functional groups is different from at least oneother functional group. Those having ordinary skill in the art, in lightof the present teachings, can determine which functional groups are mostsuitable to be included in the hydrophilic copolymer. Some non-limitingexamples of functional groups that may be present in a hydrophiliccopolymer to covalently link the latter to a magnetic bead includeamino, hydroxyl, carboxyl, N-hydroxysuccinimide (NHS) and other reactiveesters, e.g., ester of pentafluorophenol, maleimide, epoxy, aldehyde,ketone, cyanuryl, pyrrolidinedione, alkynes or azides. Covalent bondingof a hydrophilic copolymer to a magnetic bead may be via a variety oflinks, such as via thiol ether, an amide or a secondary amino link, orvia a urea or urethane.

Some non-limiting examples of functional groups that may be present in ahydrophilic copolymer to link the latter to a carbohydrate (i.e., toform glycoconjugate(s) between the carbohydrate(s) and the hydrophiliccopolymer) also include amino, hydroxyl, carboxyl, N-hydroxysuccinimide(NHS) and other reactive esters, e.g., ester of pentafluorophenol,maleimide, epoxy, aldehyde, ketone, cyanuryl, pyrrolidinedione, alkynesor azides.

One example of a class of hydrophilic polymers from which specifichydrophilic copolymer(s) can be selected is a class of acrylates bearingat least two functional groups. Non-limiting examples of suitableacrylates include functionalized copolymers having units derived fromacrylic acid or methacrylic acid, copolymers of hydroxyl-substitutedlower alkylacrylates and alkylmethacrylates, such as copolymers havingunits derived from 2-hydroxyalkyl acrylate or2-hydroxyalkylmethacrylate, acrylamides, methacrylamides,epoxy-acrylates, and derivatives of each of the above.

Another example of a class of hydrophilic copolymers from which aspecific hydrophilic copolymer(s) can be selected, to form compositionsof the present disclosure, is a class of poly(alkylene glycols) andalkoxy poly(alkylene glycols) bearing at least two functional groups.Non-limiting examples of such suitable poly(alkylene glycol s) includefunctionalized copolymers having units derived from ethylene glycol,propylene glycol and derivatives of both.

Non-limiting examples of other suitable hydrophilic copolymers that canbe used in compositions of the disclosure include functionalizedcopolymers having units derived from alcohols, e.g., vinyl alcohol andN-vinyl lactams, such as vinyl pyrrolidone, copolymers of methylvinylether and maleic acid, maleic anhydride polymers and copolymers, amides,styrenesulfonates, vinylsulfonic acid, vinylsulfonates, 3-hydroxybutyricacid, urethanes, ethyleneimines, polyurethane-polyether polymers, e.g.,urethane-poly(ethylene oxide) and N-vinylamides, e.g., N-vinylformamide,N-methyl-N-vinylformamide or N-methyl-N-vinylacetamide, and derivativesof each of the above.

In view of the teachings of this specification, those having ordinaryskill in the art may wish to select yet (an)other hydrophiliccopolymer(s), to form compositions of the present disclosure. Forexample, according to some embodiments, a hydrophilic copolymer used ina compositions of the present disclosure should include at least twofunctional groups, as explained above, and in addition a polymer isconsidered hydrophilic if it satisfies the hydrophilicity requirement asdescribed herein, i.e., if a copolymer has a range of water solubilityat ambient conditions from water miscible to slightly water soluble. Insome embodiments, the desired degree of hydrophilicity of a copolymer,as well as other desired properties of a copolymer, may be achieved bycombining with a copolymer some hydrophilic units (e.g., units formed byN,N-dimethylacrylamide) with some hydrophilic units (e.g., units formedby acrylic esters such as acrylic ester of N-hydroxysuccinimide orpentafluorophenyl acrylate), in a desired ratio. In some exemplaryembodiments, an example molar ratio between more hydrophilic units andless hydrophilic units can be between about 3:1 and about 28:1, such asbetween about 3:1 and about 10:1.

As described in sections above, a compositions of the present disclosuregenerally comprises at least one glycoconjugate, i.e., a carbohydrate(i.e., sugar) moiety attached to each hydrophilic copolymer bridge. Morethan one kind of sugar can be attached to a (same) magnetic bead via ahydrophilic copolymer bridges. For example, the number and the kinds ofcarbohydrates attached to each hydrophilic copolymer bridge can be thesame or can be different, in any combination.

A variety of carbohydrates may be used for the purpose of formingglycoconjugates with hydrophilic copolymer(s) in a composition of thedisclosure. Non-limiting examples of carbohydrates that can be usedinclude monosaccharides (e.g., glucose, galactose, fructose, mannose,lyxose, xylose, etc.), oligosaccharides, disaccharides (e.g., sucrose,lactose, maltose, isomaltose, lactulose, trehalose, etc.),trisaccharides, tetrasaccharides and polysaccarides (e.g., cellulose,glycan, dextrin, starches such as amylose or amylopectine, etc.), andN-modified derivatives of each.

Other examples of suitable carbohydrates include, but are not limitedto, sialic acid, amine-containing saccharides and N-acylated derivativesthereof, saccharide conjugates of cyclitols and other aglycans andsaccharide conjugates of aminocyclitols (e.g., aminoglycosides) andN-modified derivatives of each. In light of the present teachings, aperson having ordinary skill in the art, can selected a specificcarbohydrate that is most appropriate and suitable depending, interalia, on the end use of a composition of the disclosure.

Since microbial cellular surfaces comprise a variety of complexcarbohydrate structures, such as glycoproteins, glycolipids,glycosaminoglycans, and proteoglycans compositions of the disclosure canbe made using the methods described herein to target specifically anyparticular microbe based on a particular surface moiety present on amicrobe.

Several example methods for forming compositions are described in thereaction schemes described here. In one embodiment shown in ReactionScheme I below, a hydrophilic copolymer scaffold (or bridge) can beformed by copolymer 3 prepared by copolymerization of acrylic ester ofNHS 1 with N,N-dimethylacrylamide 2 as shown.

Using N,N-dimethylacrylamide (i.e., comonomer 2) imparts sufficienthydrophilicity to mobile copolymer scaffold 3, resulting in thereduction of non-specific adsorption of proteins and other hydrophobicbiomolecules to a composition of the disclosure shown in this reaction,while reactive acrylate ester of NHS units enable attachment ofcopolymer to magnetic beads and then enable attachment of sugar(s) tothe magnetic bead-bound polymer.

Copolymer 3 can be prepared to have various ratios between theN,N-dimethylacrylamide component and the acrylic ester of NHS component.Some examples of molar ratios between the N,N-dimethylacrylamide and theacrylic ester of NHS units in copolymer 3 may be between about 28:1 and3:1, for example, between about 10:1 and 3:1. By stoichiometric control,copolymer 3 can be immobilized onto the surfaces of magnetic beads viaamide link, by reacting at least a portion of the NHS ester groups withmost if not all the surface amino groups on a magnetic bead to form thestructure 4, which can be subsequently reacted with a sugar, such as anamino sugar, to form polyvalent magnetic bead 5.

In some embodiments, if desired, it is also possible to react a portionof NHS ester functional groups in the mobile hydrophilic copolymerscaffold 3 with an amino sugar forming a glycoconjugate prior toimmobilizing itself onto an amino magnetic bead to form 5.

In order to maximize the total number of sugar pendants and minimize thenumber of anchoring sites along a scaffold polymer chain, an acrylicacid co-monomer can be introduced as shown in Reaction Scheme II below.

As illustrated in Reaction Scheme II, a polymerizable sugar 6 can beprepared by reacting the NHS ester of acrylic acid 1 with an aminosugar. Subsequent copolymerization of 6 with N,N-dimethylacrylamide 2and acrylic acid 7 gives an acrylic copolymer 8 with pre-determinedamount of carboxylic groups for chemical immobilization onto an aminomagnetic bead to give polyvalent magnetic bead 9.

In other embodiments of the disclosure, immobilization of mobilehydrophilic copolymer scaffold onto magnetic beads can be achieved usingthe epoxy functionality on the polymer chain, as shown in ReactionScheme III.

As can be seen from Reaction Scheme III, the mobile hydrophilic polymerscaffold 12 can be prepared by copolymerization of glycidyl(meth)acrylate 10 and 2-hydroxyethyl (meth)acrylate 11. In someembodiments, the starting monomer 11 can be replaced byN,N-dimethylacrylamide 2 shown on Reaction Schemes I or II, above,acrylamide or a N-vinylamide. The sugar pendants can be incorporated byreacting an amino sugar with at least a portion, if not most, of theepoxy functional groups along the mobile hydrophilic polymer scaffold 12to yield polymer 13, which subsequently immobilizes itself onto anamine-containing magnetic bead to form a secondary amino link to affordthe final polyvalent magnetic bead 14.

In other embodiments, to immobilize mobile hydrophilic polymer scaffoldonto magnetic beads, cyanuric chloride groups on the polymer chain maybe utilized, as shown on Reaction Scheme IV.

As can be seen from Reaction Scheme IV, copolymer of 2-hydroxyethyl(meth)acrylate 17 can be prepared by copolymerization of 2-hydroxyethyl(meth)acrylate 15 with N,N-dimethyl (meth)acrylamide 16 that impartshigh hydrophilicity to the mobile scaffold after immobilization toreduce non-specific adsorption of proteins and other hydrophobicbiomolecules. Cyanuric chloride 18 can be incorporated by reacting itwith copolymer 17. The feed ratio of 18 can be adjusted such that molarratio of cyanuric chloride and 2-hydroxyethyl groups is 1:1 in copolymer19. Sugar pendants can be introduced by reacting an amino sugar with atleast a portion, if not most, of the cyanuric residues in copolymer 19to give copolymer 20. The residual cyanuric groups in 20 can be used toimmobilization onto magnetic beads to afford polyvalent magnetic beads21.

A similar approach can be used as in another embodiment shown inReaction Scheme V, where at least a portion of the hydroxyl pendantsgroups in polymer 22 can be derivatized by cyanuric chloride 18 to yieldproduct 23 that can be subsequently reacted with an amino sugar to givepolymer 24. Since the remaining two chlorides in 23 are deactivatedtoward hydroxyl group, further intermolecular reaction to form acrosslinked network may be negligible. Immobilizing product 24 onto anamino magnetic bead yields polyvalent magnetic bead 25. Polymer 22 canbe, for example, poly(2-hydroxyethyl (meth)acrylate), poly(vinylalcohol), poly(N-hydroxymethyl acrylamide), dextran or a polysaccharide.

In another embodiment, immobilization of mobile hydrophilic polymerscaffold onto magnetic beads can be via the formation of amide bonds (asin Reaction Schemes I and II above), while attachment of sugar residuesto the polymeric matrix is via copper (I) catalyzed [3+2] cycloadditionreaction between an alkyne and an alkyl azide to form a 1,2,3-triazole,commonly known in the art as “Click” chemistry. Such a method isschematically illustrated as depicted on Reaction Scheme VI.

The Click approach allows putting alkyne groups in place either prior to(conversion of 3 to 26 on reaction Scheme VI), or after (4 to 27), theattachment of the polymer to magnetic beads allowing optimization offunctional group stoichiometry and density on the surface of magneticbeads. Also, alkyne groups will be chemically inert during otherchemical manipulations. The residual unreacted alkyne groups after sugarcoupling are uncharged and stable compared to unreacted NHS-esters orepoxides.

In yet another embodiment shown in Reaction Scheme VII, glycosylattachment of azidopropyl to saccharides can be used. Thus, for example,1-(3-azidopropyl)-D-galactose (32) can be prepared frompenta-O-acetyl-D-galactose (29) via glycosylation oftrichloroacetimidate derivative (30) and then utilized in Clickchemistry for the preparation of galactose linked to polymer-modifiedmagnetic beads (product 28, Reaction Scheme VI).

Magnetic beads having surface glycoconjugates obtained according toembodiments of the present disclosure can be used for a variety ofpurposes, such as, but not limited to, to methods of capturing/detectingmicrobial cells from samples. Methods may also comprise steps ofenrichment of microbial cells captured, culturing the captured cells,lysis of captured cells to extract cellular nucleic acids and/orproteins, amplification of isolated nucleic acids from capturedmicrobial cells (TaqMan® Probe PCR), hybridization and/or sequencing forspecific identification of microbial cell types.

Various methods of using magnetic beads having surface glycoconjugatesobtained according to embodiments of the present disclosure allow toisolate microbes (including viruses, pathogens, bacteria, fungi andspores thereof) from a medium (virtually any pathogen may be isolatedfrom almost any medium). Effective rinsing/washing to removeamplification inhibitors can be also achieved. The methods of usingmagnetic beads having surface glycoconjugates obtained according toembodiments of the present disclosure also permit to eliminate stepssuch as filtration and/or centrifugation keeping isolated nucleic acidsintact.

Magnetic beads having surface glycoconjugates obtained according toembodiments of the present disclosure may be used in all applicationswhen effective and automatable capture of microorganisms from largevolume (>1 mL) of complex liquid sample is required. Such applicationsinclude, but are not limited to, food pathogens detection, bioburdendetection, biothreats detection and clinical applications.

The present disclosure also describes methods for capturing a populationof microorganisms from a biological sample, comprising: combining acomposition of the disclosure comprising: a paramagnetic bead; aplurality of hydrophilic copolymer bridges, each bridge being covalentlybonded to the paramagnetic bead; and a plurality of carbohydrates, eachcarbohydrate being covalently bonded to the same or differenthydrophilic copolymer bridge by forming a glycoconjugate with therespective hydrophilic copolymer, with the biological sample for a timeto form composition-microorganism complexes; separating thecomposition-microorganism complexes from the sample under a magneticfield; and collecting the captured microorganisms, wherein thepopulation of microorganisms has binding specificity for thecarbohydrate.

A variety of microorganisms may be detected and/or captured usingcompositions of the disclosure such as bacteria including gram negativeand gram positive species; viruses, fungi, and spores thereof.

In some embodiments of the method of capturing microorganisms, thepopulation of microorganisms captured is a subpopulation ofmicroorganisms present in the biological sample.

In some embodiments of the method, the carbohydrate is mannose and thepopulation of microorganisms has binding specificity for mannose. Forexample, a microbe may have a ligand on its surface that is operable tobind mannose moieties of a composition.

A method of capturing microorganisms of the disclosure may additionallycomprising downstream steps for detection and identification of themicrobe. Such steps in non-limiting examples may comprise one or more ofthe following steps: performing a nucleic acid extraction on thecaptured microorganisms; Real-Time PCR analysis, culturing the capturedmicroorganisms to increase the number of microorganisms for furtheranalysis, DNA sequencing, immunoassays and the like.

In one exemplary non-limited embodiment shown schematically on FIG. 4,one process of using polyvalent magnetic beads of the present disclosureto capture, enrich, and identify pathogenic microorganism may beillustrated as follows:

(1) the polyvalent magnet beads may be added into a biological sampleand incubated, followed by

(2) separating the magnetic beads under a magnetic field;

(3) washing the magnetic beads to rid of debris and non-pathogenicmaterials; and

(4) dividing the captured pathogenic microorganism for DNAextraction/Real-Time PCR, plating for MicroSEQ ID analysis, andculturing for further analysis.

In contrast to the “monovalent” binding of a pathogen cell onto acarbohydrate-modified magnetic bead as shown in FIG. 1 and described inpublished U.S. Patent Application No. 2009/0186346A1, which has weakaffinity, efficiency and binding strength, compositions of the presentdisclosure provide “polyvalent” binding that is able to withstandrepetitive washing and rinsing to remove debris and undesirablematerials from a biological sample from whichcapture/separation/detection of a microbe is desired.

EXAMPLES

The disclosure will be further described by the following examples,which are intended to be purely exemplary of the disclosure and not tolimit its scope in any way. Where cited NMR chemical shifts are in ppm(δ) and relative to the position of the solvent peak.

Example 1 Preparation of Copolymer of N,N-Dimethylacrylamide and AcrylicAcid Ester of N-hydroxysuccinimde (First Ratio Between Monomers)

Polymerization was carried out in a 250-mL round bottom glass flask withthree 14/20 ground glass joints, equipped with a half-inch magnetic stirbar, a water-cooled condenser connected to the first joint, and a rubberseptum with a glass bleeding tube for dry argon purging in the secondjoint. The water-cooled condenser was equipped with a rubber septum witha 10-gauge stainless steel syringe needle for venting into a mineral oilbubbler. A ground glass stopper was placed in the third joint. Allreagents were obtained from Sigma-Aldrich and were used as receivedexcept for N,N-dimethylacrylamide that had been vacuum distilled priorto use.

The reaction flask was charged with 100 mL of anhydrous acetonitrile,4.0371 g (40.725 mmole) of N,N-dimethylacrylamide, 0.2473 g (1.462mmole) of acrylic acid ester of N-hydroxysuccinimde and 0.0306 g (0.123mmole) of azobis(2,4-dimethylvaleronitrile). The molar ratio of theN,N-dimethylacrylamide and acrylic acid ester was 28:1. Under constant150 rpm stirring at ambient temperature, the mixture was purged bybubbling ultra pure argon for 60 minutes at a flow rate of 60 mL/min.The reaction flask was then immersed in an oil bath at 55° C. andstirred at 150 rpm for 16 hours under argon atmosphere. The reactionsolution was cooled to ambient temperature under argon atmosphere priorto be used for conjugating carbohydrate molecules onto magnetic beadshaving surface amino groups.

Example 2 Preparation of Copolymer of N,N-Dimethylacrylamide and AcrylicAcid Ester of N-hydroxysuccinimde (Second Ratio Between Monomers)

The experimental setup and polymerization conditions were the same asdescribed in Example 1. The reaction flask was charged with 100 mL ofanhydrous acetonitrile, 3.5192 g (35.501 mmole) ofN,N-dimethylacrylamide, 1.9566 g (11.571 mmole) of acrylic acid ester ofN-hydroxysuccinimde, and 0.0343 g (0.128 mmole) ofazobis(2,4-dimethylvaleronitrile). The molar ratio of theN,N-dimethylacrylamide and acrylic acid ester was 3:1.

Example 3 Preparation of Copolymer of N,N-Dimethylacrylamide andPentafluorophenyl Acrylate (First Ratio Between Monomers)

The experimental setup and polymerization conditions were the same asdescribed in Example 1. All reagents were obtained from Sigma-Aldrichexcept for pentafluorophenyl acrylate (95% pure) which was obtained fromPolysciences. N,N-dimethylacrylamide was vacuum distilled prior to use.The reaction flask was charged with 50 mL of anhydrous acetonitrile,3.0728 g (30.997 mmole) of N,N-dimethylacrylamide, 1.7538 g (7.366mmole) of pentafluorophenyl acrylate, and 0.0315 g (0.127 mmole) ofazobis(2,4-dimethylvaleronitrile). The molar ratio of theN,N-dimethylacrylamide and pentafluorophenyl acrylate was 4.2:1.

Example 4 Preparation of Copolymer of N,N-Dimethylacrylamide andPentafluorophenyl Acrylate (Second Ratio Between Monomers)

The experimental setup and polymerization conditions were the same asdescribed in Example 3. The reaction flask was charged with 50 mL ofanhydrous acetonitrile, 3.1147 g (31.421 mmole) ofN,N-dimethylacrylamide, 2.5027 g (10.511 mmole) of pentafluorophenylacrylate, and 0.0348 g (0.140 mmole) ofazobis(2,4-dimethylvaleronitrile). The molar ratio of theN,N-dimethylacrylamide and pentafluorophenyl acrylate was 3:1. Therelative incorporation of the two acrylate monomers was determined byNMR by the method of Eberhardt et al, Eur. Polymer J., 41, 1569-75(2005)).

A crude reaction solution of the copolymer in acetonitrile, nominallycontaining 500 mg of dissolved polymer, was evaporated and dried undervacuum. The residue was taken up in dry benzene (5 mL), and then addeddropwise to a flask of rapidly stirring hexane (50 mL). After severalminutes the flask was placed in the freezer overnight, whereafter a finesolid material settled out. The supernatant was decanted and the residuewas rinsed in-situ with hexane, and then re-precipitated frombenzene/hexane as before. After vacuum drying, 409 mg of the polymer wasobtained as an amorphous white solid with faint odor of acrylates.

By ¹H-NMR (400 MHz, ACN-d3) the polymer is essentially free ofunpolymerized acrylates. ¹³C-NMR indicates three carbonyl signals (δ171.1, 174.5, 175.2 ppm) in an approximately 4:1:1 ratio, correspondingrespectively to dimethylamide, pentafluorophenyl ester, and freecarboxylic acid functionality). ¹⁹F-NMR shows three broadened signals (δ−164.2, −159.8, −154.1 ppm) for the pentafluorophenyl ester in a 2:1:2ratio.

Example 5 Preparation of Copolymer of N,N-Dimethylacrylamide andPentafluorophenyl Acrylate (Third Ratio Between Monomers)

The experimental setup and polymerization conditions were the same asdescribed in Example 3. The reaction flask was charged with 50 mL ofanhydrous acetonitrile, 1.0423 g (10.514 mmole) ofN,N-dimethylacrylamide, 2.3924 g (10.048 mmole) of pentafluorophenylacrylate, and 0.0169 g (0.068 mmole) ofazobis(2,4-dimethylvaleronitrile). The molar ratio of theN,N-dimethylacrylamide and pentafluorophenyl acrylate was 1.1:1.

Example 6 Synthesis of 1-(4-Aminobutyl)-β-D-Mannose (Scheme VIII)

The synthesis of the title compound 37 was conducted as shown on theReaction Scheme VIII

4-(N-Benzyloxycarbonylamino)butan-1-ol (33, Reaction Scheme VIII) wasprepared by the method of Boseggia et al. (J. Am. Chem. Soc., 126,4543-9 (2004)). 2,3,4,6-tetra-O-acetyl-D-mannosyl trichloroacetimidate(34) was prepared by adapting the method of Cheng et al. (J. Med. Chem.,48, 645-52 (2005)), whereby, commercially available (Sigma) D-mannosepentaacetate (12.6 g, 32.4 mmol) in DMF (60 mL) was warmed to 60° C.under Ar. To this was added a suspension of hydrazine acetate salt (3.58g, 38.9 mmol) in DMF (15 mL) and the mixture was stirred overnight atambient temperature. The reaction was diluted into ethyl acetate(EtOAc), washed with water several times, then dried to afford2,3,4,6-tetra-O-acetyl-D-mannose (6.85 g, 61%).

2,3,4,6-tetra-O-acetyl-D-mannose so obtained was dissolved in drydichloromethane (DCM) (100 mL) and chilled in an ice bath, and to thiswas added trichloroacetonitrile (27.4 g, 190 mmol), followed by dropwiseaddition of an ice-cold solution of 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) (6.08 g, 40 mmol) in DCM (50 mL). The reaction was stirred to roomtemperature, then concentrated under vacuum to give a viscous yellow oilwhich was purified by silica column chromatography (hexane-EtOAc, 100:0to 1:2) to give 4.48 g pure trichloroacetimidate (34) (28% overall yieldfrom D-mannose pentaacetate).

¹H-NMR (CDCl₃): δ 8.78 (s, 1H), 6.27 (d, J=1.8 Hz), 5.46 (s, 1H), 5.39(m, 3H), 4.19 (m, 3H), 2.19 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.00(s, 3H). ¹³C-NMR (CDCl₃): δ 170.66, 169.89, 169.81, 169.70, 159.73,94.52, 71.22, 68.84, 67.88, 65.38, 62.06, 21.02, 20.85, 20.77, 20.69.

Next, protected mannosyl aminoglycoside (35) was prepared by reaction ofa solution of carbobenzyloxy(Cbz)-aminoalcohol (33) (0.54 g, 2.4 mmol)and trichloroacetimidate (34) (0.79 g, 1.6 mmol) in dry DCM (12 mL)containing 100 mg of 4 A molecular sieves, in a dry-ice bath(temperature below −30 C), with dropwise addition of trimethylsilyltrifluoromethanesulfonate (also known as TMS-triflate) (290 μL, 1.6mmol) under Ar. The reaction was stirred to ambient temperatureovernight, then diluted with DCM and washed successively with aq.bicarbonate, water, and saturated NaCl. After evaporation and drying, awhite residue was obtained which was purified by silica column(hexane-EtOAc, 90:10 to 2:1) to give 372 mg (0.67 mmol, 42%) of productcompound (35) as an amorphous white solid. MS (ESI): [M+H]+=554.4.

Next, tetraacetate (35) (0.89 g, 1.6 mmol) was dissolved in methanol (40mL) and treated with 25% sodium methoxide in methanol (1.45 mL, 4equivalents) for 3 hrs, then the mixture was partially concentrated,poured into cold water, and acetic acid was added to pH 5. The solutionwas lyophilized to give 1-(4-N-Cbz-aminobutyl)-β-D-mannose (36) (0.61 g,100%). MS (ESI): [M+Na]+=408.2. This material was dissolved in ethanol(40 mL) containing acetic acid (50 μL), charged with 5% Pd—C catalyst(100 mg) and hydrogenated overnight under 1 atm pressure toquantitatively remove the Cbz protecting group, to give1-(4-aminobutyl)-β-D-mannose (37) as the acetate salt (0.49 g, 100%).¹³C-NMR (MeOH-d4): δ 101.65, 74.71, 72.47, 72.09, 68.41, 67.92, 62.43,44.96, 27.71, 27.61. The material was sufficiently pure for subsequentuse.

Example 7 Preparation of Mannose-M270 Beads

A well-dispersed suspension of 100 μL Dynabeads M-270 Amine magneticmicrospheres (nominally 60M beads, in supplied buffer) was pipetted intoa 1.5 mL centrifuge tube. Beads were separated magnetically, the bufferwas aspirated, then beads were washed with 100 mM HEPES pH 7.0 (2×150μL), and beads were aspirated and left moist in the tube. Reactivepolymer DMA-PFP 3:1, 180 mg, was dissolved in acetonitrile (500 μL),then 100 mM HEPES pH 8.0 was added (33 μL), and then the polymersolution was added to the tube containing the beads. The mixture wasagitated by rapid vortexing until beads were well-dispersed, then thetube was placed in a heated vortexing mixer (35° C., 1400 rpm) for 3hrs, with occasional removal of the tube and vigorous vortexing to keepbeads well-dispersed. The bead suspension was magnetically separated,beads were washed with 100 mM HEPES pH 7 (3×300 μL), then polymer-coatedbeads were drained.

In another tube was placed a solution of 1-β-(4-aminobutyl)-D-mannoseprepared as described in Example 6 (75 mg, 0.30 mmol) in 100 mM HEPES pH8.0 (pH checked by meter). The amino-saccharide solution was thenintroduced and the beads were re-suspended, dispersed, and reacted at35° C./1400 rpm. After 2 hrs, the beads were separated, washed withHEPES pH 7 (3×300 μL), then resuspended in buffer (500 μL) and treatedwith a solution of 40% aq. dimethylamine (50 μL) to cap any remainingreactive esters. After vortexing for several minutes, the beads weredrained and washed with 100 mM HEPES pH 7 (3×300 μL). After draining thebeads were re-suspended and stored in 300 uL of the same buffer.

Example 8 Recovery of FimH+ Containing Bacteria by Polyvalent MannoseBead

To demonstrate that polyvalent glycoconjugate magnetic beads madeaccording to embodiments of the present disclosure can be used torecover specific microbes from solution, mannose was coupled to acopolymer of N,N-dimethylacrylamide and acrylic ester of NHS conjugatedto a magnetic bead. The molar ratio between the N,N′-dimethylacrylamideunits and the acrylic ester of NHS units was between 3:1 and 4:1. Thisbead was used to recover bacteria previously shown to bind to mannosecontaining substrates. The mannose binding E. coli K-12 strain KB54 andE. coli AAEC356 were used (J. Biol. Chem., 272, 17880-17886 (1997) andJ. Bact. 175, 4335-4344 (1993)). The KB54 strain expresses large numbersof mannose binding type 1 fimbriae from a plasmid and is known to have ahigh affinity for mono-mannose coupled BSA. The E. coli strain AAEC356constitutively expresses type 1 fimbriae and is known to bind tosurfaces coupled with mannose containing RNase B even after rigorouswashes.

The bacterial stain KB54 was obtained from E. V. Sokurenko at theUniversity of Washington, and the strain AAEC356 from I. C. Blomfield atthe University of Kent. KB54 cells were grown over night at 37° C.without shaking in Brain Heart Infusion broth containing 100 μg/mlampicillin and 25 μg/ml chloramphenicol (Sigma). AAEC356, E. coli0157:H7, Listeria monocytogenes, and Salmonella typhimurium were alsogrown overnight at 37° C. without shaking in Brain Heart Infusion broth,but without supplemental antibiotics.

Prior to recovery of bacteria with polyvalent glycoconjugate beads,cells were diluted 1×10⁶ fold with binding buffer (phosphate bufferedsaline (Invitrogen) containing 1 mM calcium chloride, 1 mM magnesiumchloride, and 0.05% Tween20 (Sigma)) to approximately 1,000 cells/ml. Torecover the bacteria, 1 ml of diluted cells was added to 0.3 mg of thebeads in a 1.5 ml Eppendorf microcentrifuge tube. Beads were incubatedwith the cells at room temperature with gentle mixing by rotation for 30min. After 30 min, the tubes were placed in a Dynal MPC-S rackcontaining its magnet (Invitrogen). After magnetic separation (˜3 min)the supernate was removed, and 100 μl of Rapid Spin Lysis Solution wasadded (Applied Biosystems).

To determine the value of 100% recovery of cells, 5 μl of a 5,000 folddilution of the overnight culture was added to 95 μl Rapid Spin LysisSolution and 0.3 mg beads. Listeria containing samples were adjusted to2 U/ul Protease K and heated at 56° C. for 30 min. All the tubes wereheated for 10 min at 95° C. and spun for 3 min at maximum rpm in anEppendorf microcentrifuge. For E. coli strains, supernate (2 μl) wasadded to EMMv2 Master Mix (18 μl) containing E. coli Taqman primers. Themixture was thermocycled on the ABI 7500 following the 7500 standardprotocol. Before addition of heated E. coli 0157:H7, Listeria orSalmonella samples to lyophilized RT-PCR reagents, heat treated sampleswere diluted 1:5 with water. The amount of 30 μl of sample was added tothe MicroSeq lyophilized RT-PCR reagents (Applied Biosystems).

It was determined that the polyvalent mannose bead recovers E. coli type1 fimbriae expressing strain cells, but not E. coli 0157:H7, Listeria orSalmonella. Briefly, as described above, cells were incubated withcontrol magnetic beads (beads coupled with polymer only) and separatelywith polyvalent mannose beads (beads coupled with polymer and thencoupled with mannose). The beads were magnetically drawn to the bottomof a tube and the supernate containing unbound cells was removed. Thecells captured by the beads were quantified by Real-Time PCR. Theresults with AAEC356 cells are shown on FIG. 5, and demonstrate thatbacteria can be efficiently recovered by polyvalent mannose magneticbeads.

In FIG. 5, the results with the Taqman probe against E. coli (E.C.probe) are shown by the bars on the left hand side in each pair andresults with the internal control probe (IPC) against a plasmid added toall samples are shown by the bars on the right hand side. The “avg CT”or average cycle threshold is the thermocycle were the fluorescence issignificantly above background. The “avg CT” represents four values—twoPCR reaction duplicates of two sample replicates. “Recovered” representsRT-PCR reactions with cells recovered by the beads, whereas “Dir add”refers to a known number of cells equal to 100% recovery by the beadsadded directly to the RT-PCR reaction. Cell number (Cell#) equal to zerocontrols were included to detect any contamination of the beads with E.coli. prior to the addition of AAEC356 cells.“Cntrl” represents controlbeads coupled with copolymer only (i.e., the above-mentioned copolymerof N,N′-dimethylacrylamide and acrylic ester of NHS having the molarratio between the N,N′-dimethylacrylamide units and the acrylic ester ofNHS units between 3:1 and 4:1), and “Mann1” and “Mann2” represent beadswhere the mannose was reacted with the polymer for 30 and 180 minrespectively.

The 80-100% recovery of AAEC356 cells by the polyvalent mannose beadswas determined by comparing the Ct of samples containing recovered cellswith the Ct obtained from cells added directly to the RT-PCR reaction(Table 1). The control bead did not recover any cells. The threepathogens did not show an affinity for mannose since the recoveries withthe control and mannose-beads were similar (Table 1). Data was collectedand analyzed as described for AAEC356 on FIG. 5.

TABLE 1 % Average Recovery Bacteria Control Bead Mannose Bead 1 MannoseBead 2 E. coli AAEC356 <1 90 ± 10 100 E. coli KB54 <4 40 ± 20 40 ± 20 E.coli 0157: H7 <1 <1 <1 Listeria 12 ± 6 12 ± 6  <1 Salmonella   4 2 ± 1 3

The failure to recover the pathogens tested with the polyvalent mannosebeads could be due to the particular strains of pathogens used notexpressing any or sufficient mannose binding fimbriae to capture thelevel of mannose currently coupled to the bead. Some E. coli 0157:H7 andSalmonella typhimurium strains do not express type 1 fimbriae, and thesefimbriae are not noted in Listeria.

Although only a few embodiments have been described in detail andexemplified above, those having ordinary skill in the art will clearlyunderstand that many modifications are possible in the describedembodiments without departing from the teachings thereof. All suchmodifications are intended to be encompassed within the followingclaims.

What is claimed is:
 1. A composition, comprising: (a) a paramagneticbead; (b) a plurality of hydrophilic copolymer bridges, each bridgebeing covalently bonded to the paramagnetic bead; and (c) a plurality ofcarbohydrates, each carbohydrate being covalently bonded to the same ordifferent hydrophilic copolymer bridge by forming a glycoconjugate withthe respective hydrophilic copolymer.
 2. The composition of claim 1,having the structure:MB-(HP)_(n)-(S)_(m), wherein MB is a magnetic bead; HP is hydrophilicpolymer bridge; S is a carbohydrate; and each of n and m is an integer,wherein n≧1 and m≧1, with the further proviso that if n=1, then m≧2. 3.The composition of claim 1, wherein each hydrophilic polymer bridge isbonded to the paramagnetic bead via a link formed by an amino group. 4.The composition of claim 1, wherein the paramagnetic bead is DYNABEADS®.5. The composition of claim 1, wherein each hydrophilic copolymer is thesame.
 6. The composition of claim 1, wherein at least one hydrophiliccopolymer is different from at least one other hydrophilic copolymer. 7.The composition of claim 1, wherein the hydrophilic copolymer bridgesare formed of functionalized hydrophilic copolymers selected from thegroup consisting of functionalized acrylates, poly(alkylene glycols),alkoxy poly(alkylene glycols), copolymers of methylvinyl ether andmaleic acid, urethanes, ethyleneimines, polyurethane-polyethercopolymers, copolymers having units derived from vinyl alcohol, N-vinyllactams, vinyl pyrrolidone, amides, maleic anhydride, styrenesulfonates,vinylsulfonic acid, vinylsulfonates, N-vinylamides or 3-hydroxybutyricacid, and derivatives thereof.
 8. The composition of claim 7, whereinthe functionalized acrylates are selected from the group consisting offunctionalized copolymers having units derived from acrylic acid,methacrylic acid, 2-hydroxyalkyl acrylate, 2-hydroxyalkylmethacrylate,acrylamides, methacrylamides, epoxy-acrylates, and derivatives thereof.9. The composition of claim 7, wherein each of the functionalizedhydrophilic copolymers includes a plurality of functional groups,comprising: (a) a first functional group to form a covalent linkagebetween the magnetic bead and the respective hydrophilic copolymerbridge; and (b) at least one second functional group, to attach at leastone carbohydrate to the hydrophilic copolymer by formingglycoconjugate(s) between the carbohydrate(s) and the hydrophiliccopolymer.
 10. The composition of claim 9, wherein in the plurality offunctional groups the functional groups are the same.
 11. Thecomposition of claim 9, wherein in the plurality of functional groups atleast one functional group is different from at least one otherfunctional group.
 12. The composition of claim 9, wherein the functionalgroups are independently selected from the group consisting of an amino,hydroxyl, carboxyl, N-hydroxysuccinimide, an ester of pentafluorophenol,maleimide, an epoxy, an aldehyde, a ketone, a cyanuryl, apyrrolidinedione, an alkyne and an azide.
 13. The composition of claim1, wherein the carbohydrates comprise monosaccharides, disaccharides,trisaccharides, tetrasaccharides, oligosaccahrides, polysaccarides orN-modified derivatives thereof.
 14. The composition of claim 13, whereinthe monosaccharides are independently selected from the group consistingof glucose, galactose, fructose, mannose, lyxose and xylose andN-modified derivatives thereof.
 15. The composition of claim 13, whereinthe disaccharides are independently selected from the group consistingof sucrose, lactose, maltose, isomaltose, lactulose, trehalose andN-modified derivatives thereof.
 16. The composition of claim 13, whereinthe polysaccharides are independently selected from the group consistingof cellulose, glycan, dextrin, amylase, amylopectine and N-modifiedderivatives thereof.
 17. The composition of claim 1, wherein thecarbohydrates are independently selected from the group consisting ofsialic acid, amine-containing saccharides, saccharide conjugates ofglycans, saccharide conjugates of aminocyclitols and N-modifiedderivatives thereof.
 18. The composition of claim 1, wherein eachcarbohydrate is the same.
 19. The composition of claim 1, wherein atleast one carbohydrate is different from at least one othercarbohydrate.
 20. The composition of claim 1, wherein each hydrophiliccopolymer has the Hildebrand solubility parameter δ of at least about 10MPa^(1/2).
 21. The composition of claim 1, wherein each hydrophiliccopolymer has the weight-averaged molecular weight of between about5,000 and about 5,000,000 Daltons.
 22. A method for fabricating aparamagnetic bead composition, the method comprising: (a) immobilizing aplurality of functionalized hydrophilic copolymers on the surface of aparamagnetic bead; and (b) bonding at least one carbohydrate to each ofat least two hydrophilic copolymers, wherein each of the functionalizedhydrophilic copolymers includes a plurality of functional groups,comprising a first functional group and at least one second functionalgroup, to thereby obtain the paramagnetic bead composition.
 23. Themethod of claim 21, wherein the step of immobilizing comprises reactingthe plurality of functionalized hydrophilic copolymers with theparamagnetic bead, wherein a covalent linkage is formed between theparamagnetic bead and each hydrophilic copolymer.
 24. The method ofclaim 21, wherein the step of bonding comprises reacting the pluralityof functionalized hydrophilic copolymers with the carbohydrate(s),wherein at least two glycoconjugates are formed between at least onecarbohydrate and the hydrophilic copolymers.
 25. The method of claim 21,wherein the hydrophilic copolymers are functionalized and selected fromthe group consisting of functionalized acrylates, poly(alkyleneglycols), alkoxy poly(alkylene glycols), copolymers of methylvinyl etherand maleic acid, urethanes, ethyleneimines, polyurethane-polyethercopolymers, and copolymers having units derived from vinyl alcohol,N-vinyl lactams, vinyl pyrrolidone, amides, maleic anhydride,styrenesulfonates, vinylsulfonic acid, vinylsulfonates or3-hydroxybutyric acid, and derivatives thereof.
 26. The method of claim25, wherein the functionalized acrylates are selected from the groupconsisting of functionalized copolymers having units derived fromacrylic acid, methacrylic acid, 2-hydroxyalkyl acrylate,2-hydroxyalkylmethacrylate, acrylamides, methacrylamides,epoxy-acrylates, and derivatives thereof.
 27. The method of claim 21,wherein in the plurality of functional groups the functional groups arethe same.
 28. The method of claim 21, wherein in the plurality offunctional groups at least one functional group is different from atleast one other functional group.
 29. The method of claim 21, whereinthe carbohydrates comprise monosaccharides, disaccharides,trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides orN-modified derivatives thereof.
 30. The method of claim 21, wherein atleast two carbohydrates are bonded and each individual carbohydrate isthe same.
 31. The method of claim 21, wherein at least two carbohydratesare bonded and at least one individual carbohydrate is different from atleast one other individual carbohydrate.
 32. A method for capturing apopulation of microorganisms from a biological sample, comprising: (a)combining the composition of claim 1 with the biological sample for atime to form composition-microorganism complexes; (b) separating thecomposition-microorganism complexes from the sample under a magneticfield; and (c) collecting the captured microorganisms, wherein thepopulation of microorganisms has binding specificity for thecarbohydrate.
 33. The method of claim 32 wherein the population ofmicroorganisms is a subpopulation of microorganisms of the biologicalsample.
 34. The method of claim 32 wherein the carbohydrate is mannoseand the population of microorganisms has binding specificity formannose.
 35. The method of claim 32, further comprising performing anucleic acid extraction and Real-Time PCR analysis of the capturedmicroorganisms.